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Asian Pacific Journal of Tropical Disease (2012)S328-S331

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Asian Pacific Journal of Tropical Disease

journal homepage:www.elsevier.com/locate/apjtd

Document heading

In vitro Antimicrobial Activity of Citrus aurantifolia and its Phytochemical

screening
Rafi khan Pathan*1, Papi Reddi Gali1, Parveen Pathan2, Tananki Gowtham1, Soujanya Pasupuleti1
Department of Pharmacy, PRIST University, Thanjavur, Tamil Nadu, India.
2Depatment of Pharmacy, NIMRA College of Pharmacy, Ibrahimpatnam, A.P, India.

ARTICLE INFO

ABSTRACT

Article history:
Received 15 June 2012
Received in revised from 5 July 2012
Accepted 7 Octoberr 2012
Available online 28 October 2012

Objective: To evaluate the antimicrobial efficacy of Citrus aurantifolia Linn (CA) against
some microorganisms - bacteria and fungus were Staphylococcus aureus, Escherichia coli,
Klebsiella pneumonia, Pseudomonas spp, Aspergillus niger, Aspergillus fumigates, Mucor spp and
Pencillium. Methods: 100 l of 10 mg CA were assessed against eight test microorganisms by
agar well Diffusion Method. Gentamicin and Ketoconazole 10 mg/ml were used as standards. A
different solvent was used to obtain CA leaf extract by using maceration technique. Results:
%yield obtained for dried leaf extract of CA with chloroform, ethanol, acetone, petroleum
ether and aqueous ethanol was approximately 15%, 18%, 09%, 11% and 24% respectively. Due
to its high yield value hydroalcoholic extract of CA was used for estimating the antimicrobial
activity and its phytochemical screening. Phytochemical screening of CA plant reveals the
presence of Alkaloids, carbohydrates, flavonoids, steroids and tannins. Conclusions: The study
demonstrates that the hydroalcoholic extract of CA leaf exhibit antibacterial activity on Klebsiella
pneumonia, Pseudomonas sp, Staphylococcus aureus and antifungal activity among Aspergillus
niger, Aspergillus fumigates, Mucor species. These recognized a good support to the use of this
plant in herbal medicine and as base for the development of new drugs and phytomedicine.

Keywords:

Citrus aurantifolia Linn

Antibacterial
Antifungal
phytomedicine
hydroalcohol

1. Introduction
Today, Traditional structure of medicine is being practiced
on many accounts. Medicinal plants are having great impact
in the field of curing diseases and as a source of medicines
for a wide variety of human ailments [1]. Several up to date
research work and practical experience have shown that using
medicinal plants is better than allopathic drugs by being safer
besides having synergistic effect. Even though large number of
medicinal plants is recognized by folklore system of medicine
but their active constituents have not yet been investigated.
Herbal medicines are prescribed widely even their biologically
active constituents are unknown because of their fewer side
effects relative low cost and effectiveness.
Human infections, particularly those involving the mucosal
and skin surfaces, constitute a major problem, especially
*Corresponding author: P.RAFI KHAN.Department of Pharmacy PRIST University,
Thanjavur, Tamil Nadu.
Phone no: 09291562935
E-mail: rafikhan930@gmail.com

in tropical and subtropical developing countries. These

microorganism will cause various diseases such like
Staphylococcus aureus (pneumonia, impetigo, cellulitis,
scalded skin syndrome, mastitis, chorioamnionitis and
neonatal sepsis), Escherichia coli (diarrhea), Klebsiella
pneumonia (pneumonia, thrombophlebitis, urinary tract
infection, cholecystitis, diarrhea, upper respiratory tract
infection, osteomyelitis, meningitis), pseudomonas (urinary
tract infections, respiratory system infections, dermatitis,
bacteremia, Pneumonia, Necrotising enterocolitis) Aspergillus
niger and Aspergillus fumigates (Aspergillosis, Allergic
Bronchopulmonary) and mucor sp (skin diseases) [2-4]
Citrus aurantifolia (CA) belong to the family Rutaceae and
are used in traditional folklore structure of medicine to
cure various diseases. C.aurantifolia is commonly called as
key lime or bitter orange and its ayurvedic properties are
Rasa, Guna and Virya. Scores of research studies have been
previously done on the plant Citrus aurantifolia that exhibits
bioactive activities for cold fevers, sore throats, sinusitis and
bronchitis, as well as helping asthma. Lime oil is mainly used
as antidepressant because it promotes refreshment to the

Rafi khan Pathan et al./Asian Paicfic Journal of Tropical Disease (2012)S328-S331

tide mind. It can be helpful for rheumatism arthritis, obesity

and cellulite and has an astringent and toning action to clear
oily skin and acne, helps with herpes, cuts and insect bites
[5-7]. During last two decades, the plant has been subjected
to extensive Phytochemical, pharmacological and clinical
investigations and many interesting findings in the areas of
Insecticidal Activity [31], Cardiac diseases, anticancer, Eye
conditions, Inflammatory Bowel Disease and Improved lung
function [32].
In present study phytochemical screening and antimicrobial
activity of hydroalcoholic extracts of leaves of Citrus
aurantifolia Linn was studied.
2. Materials and methodology
2.1 Plant Material
The leaves of CA were collected from the city of Thanjavur,
Tamil Nadu, India. The plant was identified and authenticated
(BSI/SRC/5/23/2011-12/Tech-1053) by Dr. G.V.S.Murthy, Head of
office, Tamil Nadu Agriculture University, Botanical Survey of
India, Coimbatore, Tamil Nadu, India.

2.2 Preparation of Extracts

Collected leaves were cleaned and shade air dried. The
dried leaves were pulverized by using electrical grinder and
passed through a 20-mesh sieve. A powdered leaf (50 g) was
successively extracted with Chloroform, ethanol, acetone,
petroleum ether and hydro alcohol using cold maceration
method[8]. The extraction was carried out for 72 hrs at room
temperature with mild shaking. The extracts were filtered
and concentrated at 35, and the weight of each residue was
recorded and percentage yield was calculated.

2.3 Test organisms

The test microorganisms used in this study were Bacteria:
Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia,

Pseudomonas spp. and fungus: Aspergillus niger, Aspergillus

fumigates, mucor spp and Pencillium. The test organisms
were clinical isolates and obtained from the Department of
Microbiology, PRIST University, Thanjavur, Tamil Nadu, India.
2.4 Preliminary Phytochemical Screening
Hydroalcoholic extracts of CA plant (bark, stem, roots, peel
and leaves) were subjected to preliminary phytochemical
screening for the presence or absence of various active
metabolites [9, 10].

2.5 Antibacterial activity

The antibacterial activity of the hydroalcoholic extract
was determined in accordance with the agar-well diffusion
method. The bacteria was first isolated and grown in a nutrient
broth for 18 h before use and standardize the culture to 106cfu/

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ml. Mueller-Hinton agar (OXOID) was prepared and bored the

wells into the agar using a sterile 4 mm diameter cork borer.
200 l of the standardized cell culture was spread on a MH
agar. Approximately 100 l of the hydroalcoholic extract at
10 mgml-1 were introduced into the wells, allowed to stand
at room temperature for about 2 h and then incubated at 37
. After 24 h the plates were observed for zones of inhibition.
The zone of inhibition was compared with that of control and
standard Gentamicin at a concentration of 10 gml-1.[11-22]
2.6 Antifungal activity
The fungal organisms were first isolated and allowed to grow
on a rose bengal agar (RBA) (OXOID) at 25 for 72 h. The fungi
were harvested after sporulation by pouring distilled water
on the surface of the plate and later scraped the spores with
a sterile glass rod. 100 l of the standardized fungal spore
suspension was spread on the Potassium Dextrose Agar (PDA)
using a glass spreader. Sterile 4 mm diameter of cork borer
was used to bored wells into the PDA. Approximately 100
l of 10 mg Citrus aurantifolia extract were introduced into
the wells and allowed to stand (1h) for proper diffusion of the
extract into the media. The plates were observed for zones of
inhibition after 72 h at 25 and compared with ketocanazole at
a concentration of 10 mg/ml.[23-30]

3. Results
Various parts of Citrus aurantifolia plant (leaves, stem,
roots, bark and peel) were used to estimate the presence of
active constituents. Preliminary phytochemical screening of
Hydroalcoholic extracts of Citrus aurantifolia plant was shown
in Table 1.
The leaves was extracted by using different solvents such
like Chloroform, ethanol, Acetone, Petroleum ether and
hydroalcohol and the obtained %yield value was 15%, 18%, 09%,
11% and 24% respectively. In this study hydroalcoholic leaf
extract of CA posses high solubility property and high % yield
value hence it is used for evaluating its antimicrobial activity.
The obtained zone of inhibition indicates that hydro CA leaves
exhibited in-vitro antibacterial activity against Gram-positive
and Gram-negative organisms. Control group represents the
diameter of sterile cork borer of 4 mm without any zone of
inhibition. All the bacterial strains established some degree
of sensitivity to the plant. Among the four organisms, the CA
extract showed a higher activity on Klebsiella pneumonia and
Staphylococcus aureus. (Table-2).
The obtained zone of inhibition indicates that CA leaves
exhibited in-vitro antifungal activity against Aspergillus niger,
Aspergillus fumigates and Mucor spp. Control group represents
the diameter of sterile cork borer of 4 mm without any zone
of inhibition. Hydro alcoholic extract of CA show antifungal
activity on Aspergillus niger, Aspergillus fumigates and Mucor
spp (Table 3). The results reveal that extracts of C.aurantifolia
were significantly effective against Mucor spp.

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Rafi khan Pathan et al./Asian Paicfic Journal of Tropical Disease (2012)S328-S331

Table 1
phytochemical screening of Hydroalcoholic extract of Citrus aurantifolia Plant.
Phytochemical Test
Carbohydrates ( Molischs test)
Proteins (Trichloroacetic acid test)
Amino acids (Ninhydrine test)
Alkaloids ( Dragendorff,s reagent)
Flavonoids (Shinoda test)
Steroids (Libermann-burchard test)
Triterpenoids (salkowski test)
Tannins (Ferric chloride test)
Cholesterol

Leaves

Roots

Stem

Bark

Peel

(+) = Present (-) = Absent

Table 2

Antibacterial activity of Hydroalcoholic extract of Citrus aurantifolia leaf.

Microorganism

Gram +ve/ Gram ve

Staphylococcus aureus

Escherichia coli
Klebsiella pneumonia
Pseudomonas spp

Control
04
04
04
04

Table 3
Antifungal activity of Hydroalcoholic extract of Citrus aurantifolia leaf.
Microorganism

Aspergillus niger
Aspergillus fumigates
Mucor spp
Pencillium

Zone of Inhibition (mm)

Test samples
CA

Gentamicin

10

10

16

12
12

14
15

Control

Zone of Inhibition (mm)

Test samples
CA

Ketoconazole

04

12

17

04
04
04

4. Discussion
Now a days many research are focused on herbal medicines
and their natural compounds. In traditional systems of
medicines C itrus fruits having its own importance to
treat various human alignments. Citrus juice is used as
antidepressant, promoting resistance against various infections
and famously used for scurvy disease which is caused due to
the lack of Vitamin C. In the present study we evaluate the
antibacterial and antifungal efficacy of Citrus aurantifolia
leaves although these citrus plants are very likely to infestation
by scale insects, whitefly and aphids. A different solvent was
used to prepare CA extract by using maceration technique.
Each solvent having its own capability to soluble various active
components in it. Preliminary phytochemical screening of
Hydro alcoholic leaf extract of Citrus aurantifolia reveals the
presence of Carbohydrates, Alkaloids, Flavonoids, Steroids
and Tannins. This is well known, since tannins and saponins
are important plant metabolites which is majorly responsible
for antimicrobial activity (33). Our results shows that CA leaf
extract has antibacterial activity against Klebsiella pneumonia,
Pseudomonas sp, Staphylococcus aureus and antifungal
activity amongst Aspergillus niger, Aspergillus fumigates,
Mucor species. The percentage bacterial inhibition of CA

10
16

15
21

18

leaf extract was obtained as Staphylococcus aureus (85.7%),

Klebsiella pneumonia (80%) and Pseudomonas spp (62.5%) when
compared to standard Gentamicin. The percentage fungal
inhibition of CA leaf extract was obtained as Aspergillus niger
(66.6%), Aspergillus fumigates (70.5) and Mucor spp (76.1%) when
compared to standard Ketoconazole. Our result indicates that
Hydroalcoholic leaf extract posses strongest antibacterial
activity specifically against Staphylococcus aureus.
5. Conclusions
In the present study the results indicates that the hydro
alcoholic extracts of Citrus aurantifolia leaves possess good
antibacterial and antifungal activity, confirming the great
possible of bioactive compounds and are useful for rationalizing
the use of this plant in primary health care. The results suggest
that the extract of C.aurantifolia were significantly effective
against Mucor spp in case of fungi and showed a higher activity
on Klebsiella pneumonia and Staphylococcus aureus amongst
bacteria. In vivo information may be helpful in determining
the actual potential usefulness of this plant for the handling of
causal organisms of infectious diseases. Thus further work can
be carried on the isolation procedure for finding out the exact

Rafi khan Pathan et al./Asian Paicfic Journal of Tropical Disease (2012)S328-S331

moiety responsible for the biological activity.

Acknowledgement
The authors are gratefully thanks to Honble Chancellor Prof.
P.Murugesan, PRIST University, Thanjavur.

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